1. Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, USA
2. Sanofi Pasteur, F-69367 Lyon Cedex 07, France
3. UCB SA Pharma, Braine l'Alleud, BE-1420, Belgium
4. The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
5. Ancient DNA Laboratory, School of Biological Sciences and Biotechnology, Murdoch University, Perth, Western Australia 6150, Australia
6. Department of Anatomy and Pathology, University of Kinshasa, Kinshasa B.P. 864, Democratic Republic of the Congo
7. National Institute for Biomedical Research, National Laboratory of Public Health, Kinshasa B.P. 1197, Democratic Republic of the Congo
8. Department of Pathology, University Hospital, University of Antwerp, Antwerp B-2610, Belgium
9. Present address: Centre for Ancient Genetics, Biological Institute, University of Copenhagen, Copenhagen DK-2100, Denmark.

Correspondence to: Michael Worobey¹ Correspondence and requests for materials should be addressed to M.W. (Email: worobey@email.arizona.edu).

Abstract

Human immunodeficiency virus type 1 (HIV-1) sequences that pre-date the recognition of AIDS are critical to defining the time of origin and the timescale of virus evolution^{1, 2}. A viral sequence from 1959 (ZR59) is the oldest known HIV-1 infection¹. Other historically documented sequences, important calibration points to convert evolutionary distance into time, are lacking, however; ZR59 is the only one sampled before 1976. Here we report the amplification and characterization of viral sequences from a Bouin's-fixed paraffin-embedded lymph node biopsy specimen obtained in 1960 from an adult female in Léopoldville, Belgian Congo (now Kinshasa, Democratic Republic of the Congo (DRC)), and we use them to conduct the first comparative evolutionary genetic study of early pre-AIDS epidemic HIV-1 group M viruses. Phylogenetic analyses position this viral sequence (DRC60) closest to the ancestral node of subtype A (excluding A2). Relaxed molecular clock analyses incorporating DRC60 and ZR59 date the most recent common ancestor of the M group to near the beginning of the twentieth century. The sizeable genetic distance between DRC60 and ZR59 directly demonstrates that diversification of HIV-1 in west-central Africa occurred long before the recognized AIDS pandemic. The recovery of viral gene sequences from decades-old paraffin-embedded tissues opens the door to a detailed palaeovirological investigation of the evolutionary history of HIV-1 that is not accessible by other methods.

1. Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, USA
2. Sanofi Pasteur, F-69367 Lyon Cedex 07, France
3. UCB SA Pharma, Braine l'Alleud, BE-1420, Belgium
4. The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
5. Ancient DNA Laboratory, School of Biological Sciences and Biotechnology, Murdoch University, Perth, Western Australia 6150, Australia
6. Department of Anatomy and Pathology, University of Kinshasa, Kinshasa B.P. 864, Democratic Republic of the Congo
7. National Institute for Biomedical Research, National Laboratory of Public Health, Kinshasa B.P. 1197, Democratic Republic of the Congo
8. Department of Pathology, University Hospital, University of Antwerp, Antwerp B-2610, Belgium
9. Present address: Centre for Ancient Genetics, Biological Institute, University of Copenhagen, Copenhagen DK-2100, Denmark.

Correspondence to: Michael Worobey¹ Correspondence and requests for materials should be addressed to M.W. (Email: worobey@email.arizona.edu).

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